双调控溶瘤腺病毒介导超抗原SEA基因靶向膀胱肿瘤表达

目的 构建携带SEA基因的选择性增殖腺病毒,体外实验观察SEA基因的表达及其刺激淋巴细胞对肿瘤的杀伤功能.方法 构建一种由端粒酶(hTERT)和缺氧反应元件(HIF)双重启动的携带SEA的选择性增殖腺病毒,通过逆转录-聚合酶链反应(RT-PCR)检测SEA在肿瘤细胞内mRNA表达,免疫荧光定位SEA表达于肿瘤细胞,Western blot测定蛋白表达,显微镜下动态观测淋巴细胞与肿瘤细胞共培养.酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-4分泌.结果 琼脂糖电泳252 bp处可见清晰条带;免疫荧光及Western blot用考马斯亮蓝染色显示在27 kDa附近可见蛋白清晰表达;淋巴细胞与肿瘤细胞共培养12、24、48 h实验组肿瘤细胞明显少于对照组,ELISA检测IL-4分泌量实验组均高于对照组(t=2.585 P＜0.05).结论 携带SEA基因的选择性腺病毒成功构建并表达目的基因,证明对肿瘤细胞的杀伤作用.     

肿瘤特异性启动子在溶瘤腺病毒治疗泌尿系肿瘤中的研究进展

溶瘤腺病毒(OA),也称为条件增殖型腺病毒,是经基因改造,只在肿瘤细胞中特异增殖的新一代腺病毒载体,此载体对正常细胞无影响。其能感染特定类型肿瘤细胞,通过复制、增殖、裂解并杀伤肿瘤细胞,同时利用其高效的转染效率携带治疗基因,以此提高抗肿瘤效果,利用肿瘤特异性启动子是构建OA的主要方法。本文概述了肿瘤特异性启动子在OA治...     

双靶向溶瘤腺病毒携带内皮抑素基因对肝癌的抑制作用

目的 构建双调控溶瘤腺病毒,携带小鼠内皮抑素基因(mE),研究其对裸鼠肝癌移植瘤的抗瘤活性.方法 以人端粒酶逆转录酶启动子(hTERT)和缺氧调控元件序列(HRE)调控腺病毒E1a和E1b基因,基因组插入mE基因,构建双调控溶瘤腺病毒CNHK500-mE;在裸鼠模型中观察CNHK500-mE对肝癌移植瘤模型的疗效.结果 CNHK500能够在hTERT阳性的肝癌细胞中增殖[24 h:(16.67±4.04)％;48 h:(65.33 ±7.02)％;P＜0.01],并介导mE高效表达;与空白对照组( 1895.80±323.37) mm3比较,CNHK500-mE和Ad-mE的抑瘤率分别为50.95％[(929.80±211.10) mm3,P＜0.01]和29.99％[(1327.23 ±319.36) mm3,p＜0.05];CNHK500-mE对癌组织间质血管的抑制作用明显强于Ad -mE(P ＜0.05).结论 将mE与溶瘤腺病毒结合,发挥病毒增殖的溶瘤作用和基因产物的血管抑制作用,表现出明显的协同抗瘤作用.     

携带超抗原SEA基因的特异性溶瘤腺病毒载体的构建和鉴定

目的 构建携带超抗原SEA基因的肿瘤特异性溶瘤腺病毒表达载体.方法 采用聚合酶链反应(PCR)技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中获得SEA全长基因序列,酶切后克隆入pCA13质粒,构建重组病毒质粒pCA13-SEA.将鉴定正确的pCA13-SEA与含有腺病毒右臂的质粒pBHGE3通过Lipofectamine 2000共转染HEK293细胞,经同源重组产生重组腺病毒Ad-SEA.Ad-SEA在293细胞中大量扩增并通过氯化铯密度梯度离心法纯化、测定其滴度.结果 克隆得到771bpSEA全长基因.经PCR扩增、酶切鉴定、序列测定证实,SEA基因成功克隆到溶瘤腺病毒载体中,可实现SEA基因的表达,且病毒滴度达6.5×109pfu/ml.结论 成功构建表达超抗原SEA基因的溶瘤腺病毒载体.     

携带IL-24的溶瘤腺病毒对裸鼠乳腺癌移植瘤的抑制作用

目的 构建携带IL-24基因的溶瘤腺病毒CNHK600-IL24,评估该病毒在裸鼠体内对乳腺癌的抑瘤能力.方法 将IL-24基因插入腺病毒穿梭载体SG502-△CR2,与5型腺病毒骨架载体pPE3共转染至293细胞,获得溶瘤腺病毒CNHK600-IL24.建立乳腺癌原位成瘤模型、尾静脉注射及左心室注射模拟转移瘤模型,通过尾静脉注射CNHK600-IL24,应用"活体内光学成像系统"动态地观察病毒的疗效.结果 CNHK600-IL24经测序及PCR鉴定正确,滴度为1.9×1010pfu/ml.乳腺癌原位成瘤模型:对照组的光子数以及肿瘤体积均明显高于CNHK600-IL24各治疗组(P＜0.05).CNHK600-IL24治疗后肿瘤组织出现明显的坏死,肿瘤细胞发生显著的凋亡,免疫组化检测可见肿瘤细胞内Hexon和IL-24的表达.尾静脉注射模拟转移瘤模型:对照组的裸鼠大部分于38 d前死亡,而CNHK600-IL24组的生存天数明显延长(P＜0.05).左心室注射模拟转移瘤模型:活体内光学成像可见对照组与治疗组之间具有明显差别.结论 高滴度的溶瘤腺病毒CNHK600-IL24对于乳腺癌具有明显的抑瘤效果.     

携带白细胞介素24基因溶瘤腺病毒对膀胱癌Biu87细胞的杀伤效应

目的 观察携带人白细胞介素(IL)24基因的溶瘤腺病毒(ZD55-IL24)对膀胱癌Biu87细胞的杀伤效应.方法 聚合酶链反应(PCR)鉴定溶瘤腺病毒ZD55-IL24并扩增、纯化和滴度测定.ZD55-IL24感染人膀胱癌Biu87细胞系,Western blot法检测腺病毒E1A和IL24蛋白的表达情况;原位末端标记法(TUNEL)检测细胞凋亡;通过结晶紫染色法检测细胞杀伤作用;噻唑蓝(MTT)法检测细胞存活情况.结果 PCR鉴定说明ZD55-IL24包含目的 基因且无野生型腺病毒污染;Western blot结果表明ZD55-IL24能在肿瘤细胞内表达E1A并高效介导IL24基因在Biu87细胞中的表达;TUNEL法显示ZD55-IL24能显著诱导Biu87细胞的凋亡,ZD55-IL24、ZD55-EGFP、Ad-IL24处理的Biu87细胞凋亡率分别为(52.3±3.2)%、(26.3±2.3)%、(32.0±3.1)%.结晶紫染色结果表明ZD55-IL24对Biu87细胞有明显的杀伤作用,MTT表明用MOI=10的ZD55-IL24、ZD55-EGFP和Ad-IL24感染Biu87细胞,4 d后存活率分别为(27.6±1.3)%、(48.7±2.5)%和(59.7±3.53)%.结论 成功获得纯化的溶瘤腺病毒ZD55-IL24,并进一步证明ZD55-IL24可在膀胱癌细胞内选择性地增殖并诱导膀胱癌Biu87细胞的凋亡,显示出良好的抗肿瘤效果.     

表达白细胞介素-18的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

目的 观察表达IL-18的溶瘤腺病毒(ZD55-IL18)对裸鼠肾癌移植瘤生长及血管形成的抑制作用.方法 荷肾癌裸鼠随机分4组,每组8只.瘤体内注射ZD55-IL18、溶瘤腺病毒ZD55-EGFP、表达IL-18的增殖缺陷腺病毒Ad-IL18及PBS,每次注射病毒7×108PFU/只,连续注射3 d.注射后第7天,每组处死3只取肿瘤组织,免疫组织化学检测肿瘤E1A、IL-18、CD34、VEGF表达及凋亡.第50天时处死动物测量肿瘤体积.结果 ZD55-IL18、ZD55-EGFP、Ad-IL18及生理盐水处理组肿瘤体积(mm3)分别为:299.7±52.9、536.8±90.3、570.3±99.0、766.1±145.8,ZD55-IL18与各组之间差异有统计学意义(P<0.01).Ad-IL18处理组肿瘤无E1A蛋白表达,ZD55-IL18处理组有大量E1A蛋白表达,表明病毒复制.ZD55-IL18处理组肿瘤IL-18表达及凋亡细胞阳性率均显著高于Ad-IL18处理组.ZD55-IL18处理组肿瘤CD34、VEGF表达均显著低于Ad-IL18处理组.结论 表达IL-18的溶瘤腺病毒ZD55-IL18比增殖缺陷腺病毒Ad-IL18具有更强的抑制肾癌生长及血管形成作用.     

荷载hTERT-siRNA的溶瘤腺病毒对人肾癌细胞增殖的抑制作用

目的 研究荷载hTERT-siRNA的溶瘤腺病毒(ZD-hTERT)对肾癌Ketr-3细胞增殖抑制作用.方法 ZD-hTERT、溶瘤腺病毒ZD-EGFP、表达hTERT-siRNA的增殖缺陷腺病毒AdhTERT感染人肾癌Ketr-3细胞.Western blot检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western blot检测hTERT表达;原位末端标记法(TUNEL)检测凋亡;噻唑蓝(MTT)比色法检测细胞存活;结晶紫染色法检测细胞毒作用.结果 感染ZD-hTERT、ZD-EGFP的Ketr-3细胞表达E1A;抑制hTERT表达作用依次为ZD-hTERT＞Ad-hTERT＞ZD-EGFP.感染ZD-hTERT、ZD-EGFP、AdhTERT的Ketr-3细胞凋亡率(%)分别为(65.9±1.4)、(26.5±2.8)、(16.3±3.2).感染ZDhTERT、ZD-EGFP、Ad-hTERT的Ketr-3细胞存活率(%)分别为(30.9±3.6)、(51.6±3.8)、(87.9±3.1),每种病毒之间差异有统计学意义(P＜0.01);对Ketr-3细胞的细胞毒作用ZD-hTERT＞ZD-EGFP＞Ad-hTERT.结论 表达hTERT-siRNA的溶瘤腺病毒具有显著的抑制肾癌Ketr-3细胞hTERT基因表达、诱导凋亡、杀伤肾癌细胞作用.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

目的 观察表达针对端粒酶逆转录酶(hTERT)基因的小干扰RNA(hTERT siRNA)的溶瘤腺病毒(ZD55-hTERT)抑制肾癌移植瘤生长作用.方法 荷肾癌裸鼠随机分4组,每组8只.瘤体内分别注射ZD55-hTERT、增殖缺陷型腺病毒(Ad-hTERT)、溶瘤腺病毒ZD55-EGFP及磷酸盐缓冲液(PBS),每次注射病毒7×108pfu/只,连续注射3 d.注射后第7天,每组处死3只取肿瘤组织,免疫组织化学检测肿瘤hTERT、E1A表达及凋亡.第50天时处死动物测量肿瘤体积.结果 ZD55-hTERT、Ad-hTERT、ZD55-EGFP及PBS处理组肿瘤体积(mm3)分别为:124.1±27.5、609.0±102.5、499.8±77.1、1552.1±206.4,ZD55-hTERT处理组与各组之间差异有统计学意义(P<0.01).Ad-hTERT处理组肿瘤无E1A表达,ZD55-hTERT处理组E1A大量表达,表明病毒复制.ZD55-hTERT处理组肿瘤hTERT表达显著低于Ad-hTERT处理组,凋亡细胞阳性率均显著高于Ad-hTERT处理组.结论 表达hTERT siRNA的溶瘤腺病毒ZD55-hTERT具有更强的抑制肾癌生长作用.     

溶瘤腺病毒的临床应用进展

溶瘤腺病毒,也称为条件复制性腺病毒(conditionally replicating adenovirus,CRAd),具有在肿瘤特异性增殖,而在正常细胞中不能进行增殖的特性.溶瘤腺病毒最近几年得到迅猛发展,目前已有溶瘤腺病毒的生物制剂应用到临床中,此外还有很多种溶瘤腺病毒处于临床试验中,应用前景广阔.本文就溶瘤腺病毒在临床的应用作一综述.     

Can the levels of nitric oxide in the urine,serum and tumor tissue be putative markers for bladder cancer?

AIM: To determine nitric oxide (NO) levels in serum, urine, tumor tissue and non-malignant adjacent tissue in bladder cancer patients compared with those in patients with a non-neoplastic genitourinary disease and to evaluate postoperative serum and urine NO change. METHODS: Samples was collected in 20 cancer and 41 control patients. Griess reagent was used for NO measurements. RESULTS: Mean age in the cancer and control groups was 64.2 +/- 9.9 and 63.7 +/- 8.9 years, respectively. Preoperatively, urinary infection incidence was not statistically different between groups. There were no significant differences in serum, urine and tissue NO levels in patients with and without infection in both groups. Hematuria was found to not affect urine NO level in the cancer group. Urine NO level was significantly higher than in controls preoperatively, decreased significantly following operation and remained stable after the third month. High serum NO values decreased at the early postoperative period; however, they re-increased in the long-term. No significant differences were observed in the third month in serum and urine NO levels between the patients with (no. 6) and without (no. 14) residual or relapsing tumor. No statistical difference was observed between NO levels in non-malignant adjacent and control tissues. However, tumor tissue NO level was significantly higher than those in the other two. There were no statistical differences in the first and third month serum and urine NO levels between patients who underwent bacillus Calmette-Guérin instillation therapy postoperatively and the patients who took chemotherapy or no further treatment. CONCLUSIONS: Both local and generalized host defense mechanisms seem to function in bladder cancer patients. Although further evaluation with a longer observation period and larger numbers of patients is necessary, results suggest that serum and urine NO levels are not putative and useful markers for bladder cancer.     

In vivo transfer of a neuronal nitric oxide synthase expression vector into the rat bladder by electroporation

OBJECTIVE: To investigate the possibility of in vivo gene transfer by attempting to transfer the neuronal nitric oxide synthase (nNOS) gene into rat bladder using electroporation. MATERIALS AND METHODS: The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of the marker genes green fluorescent protein (GFP) and luciferase, and the nNOS gene, was then injected into the subserosal space of the bladder and electroporation applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder and immunohistochemical staining used to detect nNOS; NO(x) released from isolated bladder strips was also assessed using microdialysis and high-performance liquid chromatography. RESULTS: From the luciferase assay, 45 V, 1 Hz, 50 ms and eight pulses were selected as the optimum conditions for electroporation. Bladder specimens with GFP genes injected by electroporation showed bright and numerous sites of GFP expression in the smooth muscle layer. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NO(x) released from bladder strips was significantly greater than in the control groups. CONCLUSIONS: These results suggest that electroporation is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.     

靶向前列腺癌并携带SATB1基因shRNA溶瘤腺病毒的构建

目的:构建DD3启动子调控并携带SATB1基因shRNA溶瘤腺病毒,研究其对前列腺癌的特异抗肿瘤作用。方法:以pSilencer3.1-SATB1为模板,通过PCR扩增出SATB1-shRNA表达框并克隆至pZD55载体,构建重组质粒pZD55-SATB1-shRNA。EcoRⅤ和XbaⅠ分别酶切pZD55-SATB1-shRNA和pZXC2-DD3-E1A,将目的片段连接重组,获得质粒pDD3-ZD55-SATB1,将其与腺病毒包装质粒pBHGE3共转染293细胞。PCR鉴定正确者即为溶瘤腺病毒DD3-ZD55-SATB1。扩增,纯化,测病毒滴度。结晶紫染色观察对前列腺癌细胞毒性;Western印迹检测E1A在列腺癌细胞中的表达;RT-PCR和Western印迹检测列腺癌细胞中SATB1基因沉默效果。结果:成功构建DD3启动子调控同时携带SATB1-shRNA的溶瘤腺病毒DD3-ZD55-SATB1,初步证实DD3-ZD55-SATB1复制具有高度的前列腺癌靶向性和特异的SATB1基因沉默效果。结论:成功构建的溶瘤腺病毒DD3-ZD55-SATB1为进一步体内外研究其对前列腺癌的治疗作用奠定基础。     

Inhibitory effects of nicorandil, a K ATP channel opener and a nitric oxide donor, on overactive bladder in animal models

OBJECTIVES

To investigate the effects of nicorandil, an ATP‐sensitive potassium (K_ATP) channel opener with a nitric oxide (NO) donor property, on overactive bladder (OAB) in animal models. Nicorandil is currently used clinically to treat ischaemic heart disease.

MATERIALS AND METHODS

Three animal OAB models were used: (i) C‐fibre mediated bladder overactivity by infusion of a low concentration of acetic acid (AA) into the bladder in female Wistar rats; (ii) bladder outlet obstruction (BOO) created by partial urethral obstruction in female Wistar rats; and (iii) neuronal NO synthase (nNOS) knockout (KO) mice with urinary frequency. The effects of nicorandil and KRN2391, both of which act as K_ATP channel openers and NO donors, on the OAB models were examined.

RESULTS

Cystometry showed that intravesical instillation of nicorandil and KRN2391 successfully inhibited OAB induced by intravesical instillation of AA. In the BOO model compared with untreated BOO rats, both nicorandil (1 and 3 mg/kg, orally) and KRN2391 (1 mg/kg, orally) significantly reduced the voiding frequency. Compared with wild‐type mice, nNOS KO mice had urinary frequency with no change in the total urine volume. Nicorandil (3 mg/kg, orally) and KRN2391 (1 mg/kg, orally) significantly reduced the voiding frequency in nNOS KO mice.

CONCLUSIONS

Our in vivo results show that nicorandil, a K_ATP channel opener with a NO donor property, can suppress OAB from both neurogenic and myogenic causes. Nicorandil appears to be a promising candidate for clinical use in patients with OAB.     

锌指E-盒结合同源异形盒-1在膀胱癌细胞中的表达及对肿瘤细胞侵袭的影响

目的:锌指E-盒结合同源异形盒-1(ZEB1)是上皮-间质转换的重要调控因子。本研究探讨ZEB1在膀胱癌细胞系中的表达情况,以及对膀胱癌发展与转移的影响。方法:采用RT-PCR检测膀胱癌细胞系ZEB1的表达,免疫荧光检测ZEB1蛋白表达定位;转染ZEB1siRNA后通过RT-PCR与Western Blot检测ZEB1的mRNA与蛋白表达变化;以及通过细胞侵袭实验观察ZEB1影响细胞侵袭能力的变化。结果:膀胱癌细胞系UMUC3和5637均表达ZEB1,SV-HUC-1不表达ZEB1;ZEB1蛋白定位于胞核;ZEB1敲低后,其mRNA与蛋白表达降低,膀胱癌细胞系侵袭能力降低。结论:膀胱癌细胞系UM-UC3和5637可用于ZEB1与肿瘤相关的机制研究;ZEB1促进膀胱癌细胞侵袭。